Strong Association between Vitamin D Receptor Gene and Severe Acute Respiratory Syndrome coronavirus 2 Infectious Variants

A coronavirus disease 2019 (COVID-19) disease, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has created significant concern since December 2019 worldwide. The virus is known to be highly transmissible. Heterogenic clinical features even vary more among SARS-CoV-2 variants from asymptomatic forms to severe symptoms. Previous studies revealed an association between COVID-19 and vitamin D deficiency resulting from its low levels in COVID-19 patients. To our knowledge, there is no scientific investigation that evaluates the direct association between SARS-CoV-2 variants of concern and vitamin D receptor ( VDR ) gene markers in Cyprus. Thus, the present study aimed to identify the putative impact of VDR gene polymorphisms on SARS-CoV-2 infection among different variants. The nasopharyngeal swabs were taken from a total number of 600 patients who were admitted to Near East University Hospital COVID-19 Polymerase Chain Reaction (PCR) Diagnosis Laboratory for routine SARS-CoV-2 real-time quantitative reverse transcription PCR (RT-qPCR) test. The RT-qPCR negative resulting samples were taken as control samples ( n  = 300). On the contrary, the case group consisted of patients who were SARS-CoV-2 RT-qPCR positive, infected with either SARS-CoV-2 Alpha ( n  = 100), Delta ( n  = 100), or Omicron ( n  = 100) variants. Two VDR gene polymorphisms, Taq I-rs731236 T > C and Fok I-rs10735810 C > T, were genotyped by polymerase chain reaction-restriction fragment length polymorphism. The mean age of the COVID-19 patient's ± standard deviation was 46.12 ± 12.36 and 45.25 ± 12.71 years old for the control group ( p  > 0.05). The gender distribution of the patient group was 48.3% female and 51.7% male and for the control group 43% female and 57% male ( p  > 0.05). Significant differences were observed in genotype frequencies of FokI and TaqI variants between SARS-CoV-2 patients compared to the control group ( p  < 0.005). Furthermore, the risk alleles, FokI T allele and TaqI C, were found to be statistically significant (odds ratio [OR] = 1.80, 95% confidence interval [CI] = 1.42–2.29, OR = 1.62, 95% CI = 1.27–2.05, respectively) in COVID-19 patients. The highest number of patients with wild-type genotype was found in the control group, which is 52.9% compared with 17.5% in the case group. Moreover, most of the COVID-19 patients had heterozygous/homozygous genotypes, reaching 82.5%, while 47.1% of the control group patients had heterozygous/homozygous genotypes. Our results suggested that patients with FokI and TaqI polymorphisms might tend to be more susceptible to getting infected with SARS-CoV-2. Overall, findings from this study provided evidence regarding vitamin D supplements recommendation in individuals with vitamin D deficiency/insufficiency in the peri- or post-COVID-19 pandemic.

A recent new coronavirus disease, the coronavirus disease 2019 (COVID-19) pandemic, has caused over 6.2 million deaths worldwide and still is a global health problem. 1,2 Viruses naturally adapt to environmental conditions over time. Therefore, viral variants might be raised due to mutations within the viral genome. These viral changes may allow the virus to spread more quickly and potentially trigger more serious complications. 3 The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome has changed over time due to random mutations, its own recombination errors, and survival adaptation, resulting in the emergence of genetic variants that are believed to be highly infectious. 4 During the pandemic, the World Health Organization declared SARS-CoV-2 variants of concern (VoC), which included Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), and Omicron (B.1.1.529). VoCs have a dramatically intensified attachment attraction in the receptor-binding domain (RBD)-human angiotensin-converting enzyme 2 complex due to mutations in the spike (S) protein RBD, enhancing the virus transmissibility. 5 Moreover, VoCs may modulate or even enhance their ability to replicate regardless of growing immunity in the population, which could be occurred via infection recovery or vaccines. 6 However, Alpha and Delta variants were presented to be more contagious and caused more deaths among the other viral genomic makeups. 7 The interaction between human genomic variations and the COVID-19 disease severity still is unclear. 8 Early studies indicated an association between COVID-19 disease and vitamin D deficiency. 9,10 A steroid hormone, vitamin D, is a fat-soluble molecule that plays a vital role in bone strengthening, muscles, and the general well-being of individuals. The primary biological function of vitamin D is to keep average calcium and phosphorus blood levels. 11 Vitamin D also has anti-inflammatory functions, especially in viral infections, and can act as an immune modulator. 12 Moreover, multiple studies have found that vitamin D has a wide range of effects on cell growth, differentiation, cell death, immunomodulation, and genome stability. Recent research has also identified a correlation between vitamin D and cardiovascular disease, diabetes, cancer, autoimmune diseases, and infectious diseases, which means that vitamin D deficiency is associated with a broad range of diseases. 13 Limited sun exposure, advanced age, a sedentary lifestyle, and vitamin D receptor (VDR) gene polymorphisms might be the factors linked to a greater risk of vitamin D deficiency diseases. 14 However, a recent study showed that the young Turkish Cypriots population in Northern Cyprus was found to be vitamin D deficient, despite the fact that the Island of Cyprus receives constant sunlight for almost 9 months a year, and the Mediterranean diet is rich in vitamin D-containing foods. 14,15 Moreover, the polymorphisms on the VDR gene included the most well-known TaqI-rs731236 T > C, ApaI-rs7975232 A > C, BsmI-rs1544410 G > A, and FokI-rs10735810 C > T, which were associated with secretion of vitamin D and a higher risk of chronic diseases like autoimmune diseases, type 2 diabetes, and cancer. 16,17 Studies on the associations of COVID-19 prevalence and mortality rates and genetic variability of the hosts showed that VDR gene polymorphisms may play role in modulating COVID-19 infection and might be related to the survival of the patients and COVID-19 severity. 18,19 Therefore, TaqI and FokI gene polymorphisms might be contributed to modulating the response to vitamin D supplementation as linked to the improved reaction to supplementation as well as a predisposition to chronic diseases. Thus, this study aimed to investigate the possible impact of VDR gene markers on different SARS-CoV-2 infectious variants that caused longpandemic.

Sample Collection
The nasopharyngeal swabs were taken from a total number of 600 patients who were admitted to Near East University Hospital COVID-19 Polymerase Chain Reaction (PCR) and 57% male (p > 0.05). Significant differences were observed in genotype frequencies of FokI and TaqI variants between SARS-CoV-2 patients compared to the control group (p < 0.005). Furthermore, the risk alleles, FokI T allele and TaqI C, were found to be statistically significant (odds ratio [ Diagnosis Laboratory for routine SARS-CoV-2 real-time quantitative reverse transcription PCR (RT-qPCR) test. RT-qPCR was performed for possible SARS-CoV-2 detection according to the manufacturer's guidelines (Uniplex RT-qPCR SARS-CoV-2 RT-qPCR Detection Kit, IKAS Medical, Nicosia, Northern Cyprus). The RT-qPCR negative resulting samples were taken as control samples (n ¼ 300). On the contrary, the case group consisted of patients who were SARS-CoV-2 RT-qPCR positive, infected with either SARS-CoV-2 Alpha (n ¼ 100), Delta (n ¼ 100), or Omicron (n ¼ 100) variants.

Vitamin D Receptor Gene Polymorphisms Genotyping
The human genomic DNA was extracted from volunteered SARS-CoV-2 RT-qPCR positive and negative (control) cases using Uniplex RT-qPCR SARS-CoV-2 RT-qPCR Detection Kit (IKAS Medical, Nicosia, Northern Cyprus). Two VDR gene polymorphisms (TaqI-rs731236 and FokI-rs10735810) were genotyped by polymerase chain reaction-restriction fragment length polymorphism. Genotypes were determined according to the presence and absence of the two restriction sites that were analyzed, and alleles were designated with respect to actual base change according to the Ensembl (http://www.ensembl.org/) genome browser and NCBI SNP database (https://www.ncbi.nlm.nih.gov/SNP/, dbSNP).

Statistical Analysis
SPSS software (Statistical Package for the Social Sciences, SPSS Inc, Chicago, IL, United States, and version 25) was used to perform a statistical analysis of the data. Descriptive data and genotype data of the study group were demonstrated as mean AE standard deviation (SD) or number and frequency, where applicable. The Student's t-test and Mann-Whitney U test were used to make comparisons between normal and nonnormally distributed quantitative variables. Chi-square (x 2 ) test was used to compare the genotype and allelic frequency distributions of FokI and TaqI polymorphisms between study groups. When the conditions for using the x 2 test were not accomplished, Pearson's chi-square test or Fisher's exact test were used to confirm the association of categorical variables between study groups. Hardy-Weinberg equilibrium was evaluated by Fischer's exact test. The odds ratio (OR) and 95% confidence interval (CI) were calculated using binary logistic regression analysis with codominant, dominant, additive, and recessive inheritance models. Akaike's information criterion was used to select the inheritance model. To assess group differences, the data were log-transformed to satisfy analysis of variance (ANOVA) criteria before being subjected to one-way ANOVA with Tukey's posthoc analysis. TaqI and FokI polymorphisms were assessed for their relative risks in SARS-CoV-2 variants infected individuals by estimating ORs and 95% CIs, which were considered separate outcomes. In all cases, differences were found important at p < 0.05.

Results
In this study, we investigated the allelic frequencies and genotypic distribution between VDR gene FokI rs10735810 C > T and TaqI rs731236 T > C polymorphisms among patients infected by different SARS-CoV-2 VoC and compared with SARS-CoV-2 RT-qPCR negative subjects as a control group.
The study group included 300 COVID-19 patients who were infected by the SARS-CoV-2 Delta, Alpha, and Omicron BA.1 variants and 300 noninfected subjects as a control group. The mean age of the COVID-19 patient's AE SD was 46.12 AE 12.36 and 45.25 AE 12.71 years old for the control group (p > 0.05). The gender distribution of the patient group was 48.3% female and 51.7% male and for the control group 43% female and 57% male (p > 0.05).

Allelic and Genotypic Distribution Frequency of Vitamin D Receptor FokI and TaqI Polymorphisms in the Studied Population
The genotypic distributions and allelic frequencies of FokI rs10735810 and TaqI rs731236 markers in COVID-19 patients and the control group are presented in ►Table 1. Significant differences were observed in genotype frequencies of FokI and TaqI variants between SARS-CoV-2-infected patients and the control group (p < 0.005) (►Table 1).
Thus, the highest number of patients with group 1 (wildtype genotype) was found in the control group, which is 52.9% compared with 17.5% in the case group. Moreover, most of the COVID-19 patients had group 2 (heterozygous/homozygous genotypes), reaching 82.5%, while 47.1% of the control group patients had group 2 (►Fig. 1). These results suggested that patients with FokI and TaqI polymorphisms might tend to be more susceptible to getting infected with SARS-CoV-2.

Vitamin D Receptor Gene Polymorphism Distributions among Severe Acute Respiratory Syndrome Coronavirus 2 Variants
Mutation analysis showed that FokI T allele was distributed in 69% of SARS-CoV-2 Delta variant-infected patients while 54% in SARS-CoV-2 Alpha variant infected patients. Additionally, 77% of SARS-CoV-2 Omicron BA.1 variant-infected patients carried at least one mutant allele for the same mutation (►Fig. 2A). The differences in the distribution of the FokI polymorphism between the three variant groups were found as statistically significant (p < 0.005).
Furthermore, TaqI T > C polymorphism was distributed in 67% of SARS-CoV-2 Delta variant subjects with at least one altered allele, while 60% of SARS-CoV-2 Alpha variant subjects carried at least one mutant allele. Also, 72% of the SARS-CoV-2 Omicron BA.1 variant infected patients carried at least one mutant allele (►Fig. 2B). However, the genotype distribution for TaqI rs731236 polymorphism among patients who were infected with different SARS-CoV-2 variants did not show any statistical significance.

Analysis of Vitamin D Receptor Gene Polymorphisms Based on Genetic Inheritance Models
In genetic association studies, statistical power to detect disease susceptibility loci depended on the genetic models tested. Therefore, the genotype frequencies were further analyzed by four genetic models: additive, codominant, dominant, and recessive models (►Table 2). For TaqI, a significant association between this polymorphism and increased risk of COVID-19 patients were found in all four models, co-dominant genotype (TT) versus (CC) (    pathophysiology might involve several signaling pathways and cellular components, including vitamin D2. 20 Vitamin D is an essential immune system modulator and has an antiinfective and immunomodulatory effect. 21 Recent studies indicated an association between COVID-19 disease and vitamin D deficiency, 9,10 also a study determined an interaction between pneumonia and low serum levels of 25-hydroxyvitamin D [25(OH) D]. 22 The primary end-point of the current study was to identify the putative interaction between VDR gene polymorphisms and SARS-CoV-2 infection among different VoCs. In our study, we observed significant differences in genotype frequencies of FokI and TaqI variants between SARS-CoV-2-infected patients and the control group (p < 0.005). Furthermore, the risk alleles, FokI T allele and TaqI C, were found to be statistically significant in COVID-19 patients. Also, when we grouped the studied SNPs according to genotypes (Group1: FokI TT (wild type) þ TaqI TT (wild type), Group2: FokI TC þ CC (heterozygote þ homozygote) þ TaqI TC þ CC (heterozygote þ homozygote) and analyzed in our study population, the results showed that patients with FokI and TaqI polymorphisms might tend to be more susceptible to getting infected with the SARS-CoV-2. A previous study showed that the VDR gene Fok1 TT genotype and/or T allele was associated with a greater risk of infection with enveloped viruses such as respiratory syncytial virus. 23 Moreover, a study in COVID-19 patients in the Cuban population showed evidence of an association between SARS-CoV-2 infection and the VDR gene TaqI polymorphism, 24 which also supported the findings of our study. A metaanalysis study indicated that vitamin D supplementation minimizes the chances of acute respiratory infections. 25 Another small-size study included 76 Spanish patients that were hospitalized due to COVID-19 disease and revealed that high doses of vitamin D application decreased the risk of intensive care unit admission. 26 Several studies demonstrated that taking vitamin D supplements could lower the possibility of viral infection severity as well as death. 25 Freitas et al implied that 76% of patients who died due to the COVID-19 were vitamin D deficient, whereas 59% of subjects had moderate symptoms and 64% of them had severe symptoms. 27 Another study, which included 77 older  patients in France, found that taking vitamin D supplements on a regular basis for a year prior to a COVID-19 infection was related to a less severe condition and a higher chance of survival than if no vitamin D was taken. 28 These studies support the outcomes of the current study, demonstrating a strong association between vitamin D and COVID-19 disease. However, none of the studies determined that VDR gene polymorphisms affect COVID-19 disease outcome as well as the SARS-CoV-2 variant impact on the disease and there was no scientific investigation that evaluates the association between SARS-CoV-2 VoC and VDR gene variations.
Furthermore, we investigated the putative impact of VDR gene polymorphisms on SARS-CoV-2 infection with different variants. The significant differences observed in the distribution of the FokI polymorphism between the three variant groups were found as statistically significant (p < 0.005). We supposed that the FokI polymorphism may be related to the severity of COVID-19 in patients. Unfortunately, we could not access the patients' clinical data, which is one of the limitations of our study, and could not confirm the association between the FokI polymorphism and COVID-19 severity. However, findings of a recent study by Apaydin et al implied that serum 25 (OH) D levels had no relation to COVID-19 severity or mortality. Furthermore, it was discovered that VDR polymorphisms are independently related to the severity of COVID-19 and patient survival. 29 In addition, recent Israeli research based on the first two pandemic waves, which was prior to vaccine usage, discovered a risk of having severe COVID-19 in vitamin D-deficient individuals. 30 This case-control study is one of the few studies evaluating the association between SARS-CoV-2 VoC and VDR gene variations. Previous research had mostly used case reports and investigated mainly association between vitamin D levels and COVID-19 disease.
Nonetheless, due to certain limitations, our findings must be interpreted with caution. Limitations of this study included the lack of vitamin D serum level information for the participants and the lack of access to the patients' clinical data and not including another two important VDR gene polymorphisms ApaI-rs7975232 A > C and BsmI-rs1544410 G > A. Further clinical trials and research with a broader range of data are required to confirm the results of our study and further investigate the association between SARS-CoV-2 VoC and VDR gene variations.

Conclusion
To conclude, the COVID-19 disease is still an important public health concern worldwide. This pandemic has severely harmed health care systems' ability to continue providing quality health care. As health care systems around the world struggle to meet the rising demand for COVID-19 care, sustaining preventive and therapeutic services is critical, especially for the most vulnerable populations such as children, the elderly, people with chronic illnesses, minorities, and people with disabilities.
It is very crucial to maintain preventive measures to avoid getting infected or minimize the severity of the disease by maintaining a safe distance from others and eating a wellbalanced diet to be a healthier individual with a stronger immune system.
Our results displayed important differences in VDR polymorphisms' genotype distribution among different SARS-CoV-2 variants. The findings also implied that the patients with FokI and TaqI gene polymorphisms might be more susceptible to getting infected with SARS-CoV-2 VoC and provided evidence regarding vitamin D supplements recommendation in individuals with vitamin D deficiency/insufficiency in the peri-or post-COVID-19 pandemic.

Ethical Approval
All procedures performed in this study were in accordance with the ethical standards of the institutional research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards (Approval number: YDU/202299-1451).

Data Availability Statement
The data is available upon request.